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二硫键稳定的抗bFGF人源双链抗体的构建及表达

发表时间:2013-03-15  浏览量:897  下载量:660
全部作者: 姜浩武,亢中奎,王宏,劳学军,吕卫东,唐勇,宋其芳,黄建芳,邓宁
作者单位: 暨南大学广东省分子免疫和抗体工程重点实验室;暨南大学附属第一医院肝胆外科
摘 要: 目的:构建和表达二硫键稳定的抗碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)人源性双链抗体。方法:以抗bFGF人源性单链Fv抗体(single chain Fv,ScFv)基因为模板,以PCR和重叠PCR(overlap PCR)的方法,用短的连接肽连接抗体的轻重链可变区,并将其轻重链可变区的适当部位定点突变成半胱氨酸,构建成二硫键稳定的双链抗体(disulphide stabilized diabody,ds-Diabody)。结果:测序证实ds-Diabody基因正确;Western blotting结果显示不同肽链间的二硫键能正确配对,2条肽链可成功形成二聚体;ELISA结果显示相对于ScFv,ds-Diabody能更好地与抗原bFGF进行特异性结合,并具有良好的稳定性。结论:成功构建和表达了抗bFGF人源性ds-Diabody,在ScFv的基础上改善了抗体的亲和力和稳定性,从而为bFGF抗体药物的研究奠定了基础。
关 键 词: 分子生物学;二硫键稳定的双链抗体;二硫键;哺乳动物细胞;稳定性
Title: Construction and expression of a human diabody against bFGF stabilized by disulphide
Author: JIANG Haowu, KANG Zhongkui, WANG Hong, LAO Xuejun, LV Weidong, TANG Yong, SONG Qifang, HUANG Jianfang, DENG Ning
Organization: Guangdong Province Key Laboratory of Molecular Immunology and Antibody Engineering, Jinan University; Department of Hepatobiliary Surgery, the First Affiliated Hospital, Jinan University
Abstract: Objective: To construct and express a human disulphide stabilized diabody (ds-Diabody) against basic fibroblast growth factor (bFGF). Methods: Taking anti-bFGF human-derived single chain Fv antibody (ScFv) genes as template, using the methods of PCR and overlap PCR, the ds-Diabody was constructed by means of linking the heavy and light chain variable domains of antibody with short linking peptide, together with the mutation of the amino acids to cysteine residues in suitable position of the heavy and light chain variable domains of a human ScFv against bFGF. Results: The results of sequencing showed that the gene of ds-Diabody were constructed correctly. Western blotting demonstrated that disulfide bonds between the different peptide chains could be paired correctly to successfully form a dimer of two polypeptide chains. The ELISA results showed that the ds-Diabody has a higher specific binding activity with bFGF and more stability compared with ScFv. Conclusion: The human ds-Diabody against bFGF was constructed successfully, the affinity and stability of the antibody was improved based on ScFv, which may provide a basis for drug research of bFGF antibody.
Key words: molecular biology; disulphide stabilized diabody; disulphide bond; mamalian cell; stability
发表期数: 2013年3月第5期
引用格式: 姜浩武,亢中奎,王宏,等. 二硫键稳定的抗bFGF人源双链抗体的构建及表达[J]. 中国科技论文在线精品论文,2013,6(5):396-401.
 
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